Immunoevasive Engineered Living Blood Vessels

免疫逃避工程活血管

• 批准号: 10676153
• 负责人: Elliot Chaikof
• 金额: $ 60.42万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-05 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676153
• 关键词: Ablation; Allogenic; Aneurysm; Arteries; Biochemical; Biological; Biomechanics; Blood Vessels; CD47 gene; Cell Wall; Cells; Chemicals; Collagen; Collagen Fiber; Collagen Fibril; Elastin; Elements; Endothelial Cells; Engineering; Extracellular Matrix; Failure; HLA G antigen; HLA-A gene; Homing; Human; Immune Evasion; Immune system; Immunoassay; Immunologic Deficiency Syndromes; Implant; In Vitro; Infiltration; Macrophage; Mechanics; Microfabrication; Morphology; Mus; Natural Killer Cells; Operative Surgical Procedures; Patients; Phase; Phenotype; Physiological; Process; Production; Property; Proteins; Protocols documentation; Rattus; Reporting; Research Personnel; Risk; Smooth Muscle Myocytes; Source; Stenosis; Structure; T-Lymphocyte; Techniques; Thick; Tissue Engineering; Vascular Smooth Muscle; biodegradable polymer; design; differentiation protocol; fabrication; genome editing; human pluripotent stem cell; hydrodynamic flow; immunogenicity; immunoregulation; implantation; in vivo; innovation; large scale production; manufacture; materials science; meter; programmed cell death ligand 1; reconstitution; response; scaffold; stem cell biology; stem cells; tool; vascular tissue engineering

Project Summary Recent innovations by project investigators have established an important new framework for the rapid and scalable production of engineered living blood vessels. Notably, we have designed new protocols for multiplex genome editing to generate human pluripotent stem cells (hPSCs) in which HLA-A, -B, and -C were selectively ablated, HLA class II molecules eliminated, and multiple tolerogenic factors, including HLA-G, PD-L1, and CD47 expressed. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) derived from these PSCs, using our previously reported chemically defined differentiation protocols, were protected from alloimmune rejection in vitro and in vivo. Further, we have developed new engineering approaches for the fabrication of mechanically robust, free-standing, ultrathin collagen sheets and related manufacturing tools for the scalable production of engineered living blood vessels. In this proposal, we postulate that immunoevasive blood vessels can be efficiently and rapidly manufactured using ‘hypoimmunogenic’ cells and planar extracellular matrix (ECM) scaffolds of defined composition, content, and microarchitecture. In the process, the efficacy of a variety of tolerogenic strategies will be evaluated. In this proposal we intend to: (1) Define the morphological and structural remodeling responses of an engineered living blood vessel substitute designed to mimic the microstructure of the native vessel wall. Engineered vessels will be fabricated by seeding primary human vascular wall cells on ultrathin ECM sheets consisting of collagen fibers or a collagen-elastin multilamellar composite. Biomechanical properties will be tuned in response to microstructure, and both biochemical and functional responses defined under simulated physiological conditions. Vessels will be implanted into immunodeficient SRG rats and both phenotypic stability and remodeling responses defined. (2) Generate ‘hypoimmunogenic’ vascular smooth muscle cells and endothelial cells that evade immunological rejection. ECs and SMCs will be derived from hypoimmunogenic hPSCs generated by multiplex genome editing and biological properties determined, including differentiation efficiency, functionality, absence of HLA proteins, and expression of tolerogenic factors. Angiogenic potential and vessel network formation will be assessed in vitro and in vivo. Alloreactivity will be evaluated using an in vitro panel of T cell, NK cell, and macrophage immunoassays, as well as in mice containing human immune system components. (3) Characterize the phenotypic stability, immunogenicity, and remodeling responses of immunoevasive engineered living blood vessels. Engineered vessels comprised of hypoimmunogenic cells will be produced and related biomechanical and biochemical properties characterized. We will determine the capacity of these vessels to maintain phenotypic stability after in vivo implantation in immunodeficient SRG rats. In the final phase of these studies, we will determine the ability of vessels engineered from hypoimmunogenic SMCs and ECs to evade immunological rejection in SRG rats reconstituted with elements of a human immune system.

项目摘要 项目调查人员最近的创新为快速开发建立了一个重要的新框架 以及可规模化生产工程活血管。值得注意的是，我们设计了新的协议用于 多重基因组编辑以产生人类多能干细胞(HPSCs)，其中HLA-A、-B和-C是 选择性消融，消除人类白细胞抗原II类分子，以及多种耐受因子，包括人类白细胞抗原G，PD-L1， CD47表达。由此衍生的血管平滑肌细胞(SMC)和内皮细胞(ECs) 使用我们之前报道的化学定义的分化方案的PSCs被保护不受 体外和体内的同种异体免疫排斥反应。此外，我们还开发了新的工程方法来 机械坚固的、独立的、超薄的胶原蛋白片材和相关制造工具的制造 可规模化生产工程活血管。在这项提议中，我们假设免疫回避 使用低免疫原性细胞和平面细胞外细胞可以高效、快速地制造血管 定义了组成、内容和微体系结构的矩阵(ECM)支架。在这个过程中，一种 将对各种耐受策略进行评估。在这项建议中，我们打算： (1)确定工程化活血管的形态和结构重塑反应 设计用于模拟本地血管壁的微观结构的替代品。工程船将是 将原代人血管壁细胞种植在由胶原纤维或胶原纤维组成的超薄ECM片上 一种胶原-弹性蛋白多层复合材料。生物力学性能将根据微观结构进行调整， 以及在模拟生理条件下定义的生化和功能反应。船只将被 移植到免疫缺陷的SRG大鼠体内，定义了表型稳定性和重塑反应。 (2)产生免疫原性低的血管平滑肌细胞和内皮细胞 免疫排斥反应。ECS和SMC将来自以下来源的低免疫原性hPSCs 多重基因组编辑和生物学特性的确定，包括分化效率、功能、 缺乏人类白细胞抗原蛋白，表达耐受因子。血管生成潜能与血管网络 将在体外和体内评估其形成情况。同种异体反应性将使用T细胞体外小组NK进行评估 细胞、巨噬细胞免疫分析，以及含有人类免疫系统成分的小鼠。 (3)鉴定免疫逃逸的表型稳定性、免疫原性和重塑反应。 人工制造的活血管。将生产由低免疫原性细胞组成的工程化血管 并对相关的生物力学和生化特性进行表征。我们将确定这些设备的容量 免疫缺陷SRG大鼠体内植入后维持表型稳定性的血管。在最后阶段 在这些研究中，我们将确定由低免疫原性SMC和ECs工程生成的血管的能力 用人类免疫系统的元素重组的SRG大鼠逃避免疫排斥反应。